Hsp104, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions

Self-templating amyloid forms of Sup35 constitute the yeast prion [PSI(+)]. How the protein-remodelling factor, Hsp104, collaborates with other chaperones to regulate [PSI(+)] inheritance remains poorly delineated. Here, we report how the Ssa and Ssb components of the Hsp70 chaperone system directly affect Sup35 prionogenesis and cooperate with Hsp104. We identify the ribosome-associated Ssb1:Zuo1:Ssz1 complex as a potent antagonist of Sup35 prionogenesis. The Hsp40 chaperones, Sis1 and Ydj1, preferentially interact with Sup35 oligomers and fibres compared with monomers, and facilitate Ssa1 and Ssb1 binding. Various Hsp70:Hsp40 pairs block prion nucleation by disassembling molten oligomers and binding mature oligomers. By binding fibres, Hsp70:Hsp40 pairs occlude prion recognition elements and inhibit seeded assembly. These inhibitory activities are partially relieved by the nucleotide exchange factor, Fes1. Low levels of Hsp104 stimulate prionogenesis and alleviate inhibition by some Hsp70:Hsp40 pairs. At high concentrations, Hsp104 eliminates Sup35 prions. This activity is reduced when Ssa1, or enhanced when Ssb1, is incorporated into nascent prions. These findings illuminate several facets of the chaperone interplay that underpins [PSI(+)] inheritance.     

Molecular chaperones and the assembly of the prion Sup35p, an in vitro study

The protein Sup35 from Saccharomyces cerevisiae possesses prion properties. In vivo, a high molecular weight form of Sup35p is associated to the [PSI+] factor. The continued propagation of [PSI+] is highly dependent on the expression levels of molecular chaperones from the Hsp100, 70 and 40 families; however, so far, their role in this process is unclear. We have developed a reproducible in vitro system to study the effects of molecular chaperones on the assembly of full-length Sup35p. We show that Hsp104p greatly stimulates the assembly of Sup35p into fibrils, whereas Ydj1p has inhibitory effect. Hsp82p, Ssa1p and Sis1p, individually, do not affect assembly. In contrast, Ssa1p together with either of its Hsp40 cochaperones blocks Sup35p polymerization. Furthermore, Ssa1p and Ydj1p or Sis1p can counteract the stimulatory activity of Hsp104p, by forming complexes with Sup35p oligomers, in an ATP-dependent manner. Our observations reveal the functional differences between Hsp104p and the Hsp70-40 systems in the assembly of Sup35p into fibrils and bring new insight into the mechanism by which molecular chaperones influence the propagation of [PSI+].     

Nucleotide exchange factors for Hsp70s are required for [URE3] prion propagation in Saccharomyces cerevisiae

The [URE3] and [PSI(+)] prions are infectious amyloid forms of Ure2p and Sup35p. Several chaperones influence prion propagation: Hsp104p overproduction destabilizes [PSI(+)], whereas [URE3] is sensitive to excess of Ssa1p or Ydj1p. Here, we show that overproduction of the chaperone, Sse1p, can efficiently cure [URE3]. Sse1p and Fes1p are nucleotide exchange factors for Ssa1p. Interestingly, deletion of either SSE1 or FES1 completely blocked [URE3] propagation. In addition, deletion of SSE1 also interfered with [PSI(+)] propagation.     

Antagonistic interactions between yeast [PSI(+)] and [URE3] prions and curing of [URE3] by Hsp70 protein chaperone Ssa1p but not by Ssa2p

The yeast [PSI(+)], [URE3], and [PIN(+)] genetic elements are prion forms of Sup35p, Ure2p, and Rnq1p, respectively. Overexpression of Sup35p, Ure2p, or Rnq1p leads to increased de novo appearance of [PSI(+)], [URE3], and [PIN(+)], respectively. This inducible appearance of [PSI(+)] was shown to be dependent on the presence of [PIN(+)] or [URE3] or overexpression of other yeast proteins that have stretches of polar residues similar to the prion-determining domains of the known prion proteins. In a similar manner, [PSI(+)] and [URE3] facilitate the appearance of [PIN(+)]. In contrast to these positive interactions, here we find that in the presence of [PIN(+)], [PSI(+)] and [URE3] repressed each other's propagation and de novo appearance. Elevated expression of Hsp104 and Hsp70 (Ssa2p) had little effect on these interactions, ruling out competition between the two prions for limiting amounts of these protein chaperones. In contrast, we find that constitutive overexpression of SSA1 but not SSA2 cured cells of [URE3], uncovering a specific interaction between Ssa1p and [URE3] and a functional distinction between these nearly identical Hsp70 isoforms. We also find that Hsp104 abundance, which critically affects [PSI(+)] propagation, is elevated when [URE3] is present. Our results are consistent with the notion that proteins that have a propensity to form prions may interact with heterologous prions but, as we now show, in a negative manner. Our data also suggest that differences in how [PSI(+)] and [URE3] interact with Hsp104 and Hsp70 may contribute to their antagonistic interactions.     

Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.     

Functionally redundant isoforms of a yeast Hsp70 chaperone subfamily have different antiprion effects

Why eukaryotes encode multiple Hsp70 isoforms is unclear. Saccharomyces cerevisiae Ssa1p and Ssa2p are constitutive 98% identical Hsp70's. Stress-inducible Ssa3p and Ssa4p are 80% identical to Ssa1/2p. We show Ssa1p-4p have distinct functions affecting [PSI(+)] and [URE3] prions. When expressed as the only Ssa, Ssa1p antagonized [URE3] and Ssa2p antagonized [PSI(+)]. Ssa3p and Ssa4p influenced [URE3] and [PSI(+)] somewhat differently but overall their effects paralleled those of Ssa1p and Ssa2p, respectively. Additionally, Ssa3p suppressed a prion-inhibitory effect of elevated temperature. Our previously described Ssa1-21p mutant weakens [PSI(+)] in SSA1-21 SSA2 cells and abolishes it in SSA1-21 ssa2Delta cells. To test if the same mutation affected other prions or altered Ssa2p similarly, we compared effects of a constructed Ssa2-21p mutant and Ssa1-21p on both prions. Surprisingly, [URE3] was unaffected in SSA1-21 SSA2 cells and could propagate in SSA1-21 ssa2Delta cells. Ssa2-21p impaired [URE3] considerably and weakened [PSI(+)] strongly but in a manner distinct from Ssa1-21p, highlighting functional differences between these nearly identical Hsp70's. Our data uncover exquisite functional differences among isoforms of a highly homologous cytosolic Hsp70 subfamily and point to a possibility that variations in Hsp70 function that might improve fitness under optimal conditions are also important during stress.     

URE3] prion propagation in Saccharomyces cerevisiae: requirement for chaperone Hsp104 and curing by overexpressed chaperone Ydj1p

The [URE3] nonchromosomal genetic element is an infectious form (prion) of the Ure2 protein, apparently a self-propagating amyloidosis. We find that an insertion mutation or deletion of HSP104 results in inability to propagate the [URE3] prion. Our results indicate that Hsp104 is a common factor in the maintenance of two independent yeast prions. However, overproduction of Hsp104 does not affect the stability of [URE3], in contrast to what is found for the [PSI(+)] prion, which is known to be cured by either overproduction or deficiency of Hsp104. Like Hsp104, the Hsp40 class chaperone Ydj1p, with the Hsp70 class Ssa1p, can renature proteins. We find that overproduction of Ydj1p results in a gradual complete loss of [URE3]. The involvement of protein chaperones in the propagation of [URE3] indicates a role for protein conformation in inheritance.     

Saccharomyces cerevisiae Hsp70 mutations affect [PSI+] prion propagation and cell growth differently and implicate Hsp40 and tetratricopeptide repeat cochaperones in impairment of [PSI+

We previously described an Hsp70 mutant (Ssa1-21p), altered in a conserved residue (L483W), that dominantly impairs yeast [PSI(+)] prion propagation without affecting growth. We generated new SSA1 mutations that impaired [PSI(+)] propagation and second-site mutations in SSA1-21 that restored normal propagation. Effects of mutations on growth did not correlate with [PSI(+)] phenotype, revealing differences in Hsp70 function required for growth and [PSI(+)] propagation and suggesting that Hsp70 interacts differently with [PSI(+)] prion aggregates than with other cellular substrates. Complementary suppression of altered activity between forward and suppressing mutations suggests that mutations that impair [PSI(+)] affect a similar Hsp70 function and that suppressing mutations similarly overcome this effect. All new mutations that impaired [PSI(+)] propagation were located in the ATPase domain. Locations and homology of several suppressing substitutions suggest that they weaken Hsp70's substrate-trapping conformation, implying that impairment of [PSI(+)] by forward mutations is due to altered ability of the ATPase domain to regulate substrate binding. Other suppressing mutations are in residues important for interactions with Hsp40 or TPR-containing cochaperones, suggesting that such interactions are necessary for the impairment of [PSI(+)] propagation caused by mutant Ssa1p.     

Molecular chaperones and the assembly of the prion Ure2p in vitro

The protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3]is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled full-length Ure2p and Ure2p-(94-354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3].     

N-terminal domain of yeast Hsp104 chaperone is dispensable for thermotolerance and prion propagation but necessary for curing prions by Hsp104 overexpression

Hsp104 is a hexameric protein chaperone that resolubilizes stress-damaged proteins from aggregates. Hsp104 promotes [PSI(+)] prion propagation by breaking prion aggregates, which propagate as amyloid fibers, into more numerous prion "seeds." Inactivating Hsp104 cures cells of [PSI(+)] and other amyloid-like yeast prions. Overexpressing Hsp104 also eliminates [PSI(+)], presumably by completely resolubilizing prion aggregates. Inexplicably, however, excess Hsp104 does not cure the other prions. Here we identify missense mutations in Hsp104's amino-terminal domain (NTD), which is conserved among Hsp100 proteins but whose function is unknown, that improve [PSI(+)] propagation. Hsp104Delta147, engineered to lack the NTD, supported [PSI(+)] and functioned normally in thermotolerance and protein disaggregation. Hsp104Delta147 failed to cure [PSI(+)] when overexpressed, however, implying that excess Hsp104 does not eliminate [PSI(+)] by direct dissolution of prion aggregates. Curing of [PSI(+)] by overexpressing catalytically inactive Hsp104 (Hsp104KT), which interferes with endogenous Hsp104, did not require the NTD. We further found that Hsp104 mutants defective in threading peptides through the hexamer pore had reduced ability to support [PSI(+)] in proportion to protein resolubilization defects, suggesting that [PSI(+)] propagation depends on this threading and that Hsp104 "breaks" prion aggregates by extracting protein monomers from the amyloid fibers.     

Guanidine hydrochloride inhibits the generation of prion "seeds" but not prion protein aggregation in yeast

[PSI(+)] strains of the yeast Saccharomyces cerevisiae replicate and transmit the prion form of the Sup35p protein but can be permanently cured of this property when grown in millimolar concentrations of guanidine hydrochloride (GdnHCl). GdnHCl treatment leads to the inhibition of the replication of the [PSI(+)] seeds necessary for continued [PSI(+)] propagation. Here we demonstrate that the rate of incorporation of newly synthesized Sup35p into the high-molecular-weight aggregates, diagnostic of [PSI(+)] strains, is proportional to the number of seeds in the cell, with seed number declining (and the levels of soluble Sup35p increasing) in the presence of GdnHCl. GdnHCl does not cause breakdown of preexisting Sup35p aggregates in [PSI(+)] cells. Transfer of GdnHCl-treated cells to GdnHCl-free medium reverses GdnHCl inhibition of [PSI(+)] seed replication and allows new prion seeds to be generated exponentially in the absence of ongoing protein synthesis. Following such release the [PSI(+)] seed numbers double every 20 to 22 min. Recent evidence (P. C. Ferreira, F. Ness, S. R. Edwards, B. S. Cox, and M. F. Tuite, Mol. Microbiol. 40:1357-1369, 2001; G. Jung and D. C. Masison, Curr. Microbiol. 43:7-10, 2001), together with data presented here, suggests that curing yeast prions by GdnHCl is a consequence of GdnHCl inhibition of the activity of molecular chaperone Hsp104, which in turn is essential for [PSI(+)] propagation. The kinetics of elimination of [PSI(+)] by coexpression of a dominant, ATPase-negative allele of HSP104 were similar to those observed for GdnHCl-induced elimination. Based on these and other data, we propose a two-cycle model for "prionization" of Sup35p in [PSI(+)] cells: cycle A is the GdnHCl-sensitive (Hsp104-dependent) replication of the prion seeds, while cycle B is a GdnHCl-insensitive (Hsp104-independent) process that converts these seeds to pelletable aggregates.     

J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation

Yeast prions are protein-based genetic elements capable of self-perpetuation. One such prion, [RNQ(+)], requires the J-protein Sis1, an Ssa Hsp70 co-chaperone, as well as the AAA+ ATPase, Hsp104, for its propagation. We report that, upon depletion of Sis1, as well as upon inactivation of Hsp104, Rnq1 aggregates increased in size. Subsequently, cells having large aggregates, as well as an apparently soluble pool of Rnq1, became predominant in the cell population. Newly synthesized Rnq1 localized to both aggregates and bulk cytosol, suggesting that nascent Rnq1 partitioned into pools of prion and nonprion conformations, and implying that these large aggregates were still active as seeds. Ultimately, soluble Rnq1 predominated, and the prion was lost from the population. Our data suggest a model in which J-protein:Hsp70 machinery functions in prion propagation, in conjunction with Hsp104. Together, these chaperones facilitate fragmentation of prion polymers, generating a sufficient number of seeds to allow efficient conversion of newly synthesized Rnq1 into the prion conformation.     

Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+

[PSI(+)] is a prion isoform of the yeast release factor Sup35. In some assays, the cytosolic chaperones Ssa1 and Ssb1/2 of the Hsp70 family were previously shown to exhibit "pro-[PSI(+)]" and "anti-[PSI(+)]" effects, respectively. Here, it is demonstrated for the first time that excess Ssa1 increases de novo formation of [PSI(+)] and that pro-[PSI(+)] effects of Ssa1 are shared by all other Ssa proteins. Experiments with chimeric constructs show that the peptide-binding domain is a major determinant of differences in the effects of Ssa and Ssb proteins on [PSI(+)]. Surprisingly, overproduction of either chaperone increases loss of [PSI(+)] when Sup35 is simultaneously overproduced. Excess Ssa increases both the average size of prion polymers and the proportion of monomeric Sup35 protein. Both in vivo and in vitro experiments uncover direct physical interactions between Sup35 and Hsp70 proteins. The proposed model postulates that Ssa stimulates prion formation and polymer growth by stabilizing misfolded proteins, which serve as substrates for prion conversion. In the case of very large prion aggregates, further increase in size may lead to the loss of prion activity. In contrast, Ssb either stimulates refolding into nonprion conformation or targets misfolded proteins for degradation, in this way counteracting prion formation and propagation.     

Molecular chaperone Hsp104 can promote yeast prion generation

[URE3] is an amyloid-based prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. The Ure2p of the human pathogen Candida albicans can also be a prion in S. cerevisiae. We find that overproduction of the disaggregating chaperone, Hsp104, increases the frequency of de novo [URE3] prion formation by the Ure2p of S. cerevisiae and that of C. albicans. This stimulation is strongly dependent on the presence of the [PIN(+)] prion, known from previous work to enhance [URE3] prion generation. Our data suggest that transient Hsp104 overproduction enhances prion generation through persistent effects on Rnq1 amyloid, as well as during overproduction by disassembly of amorphous Ure2 aggregates (generated during Ure2p overproduction), driving the aggregation toward the amyloid pathway. Overproduction of other major cytosolic chaperones of the Hsp70 and Hsp40 families (Ssa1p, Sse1p, and Ydj1p) inhibit prion formation, whereas another yeast Hsp40, Sis1p, modulates the effects of Hsp104p on both prion induction and prion curing in a prion-specific manner. The same factor may both enhance de novo prion generation and destabilize existing prion variants, suggesting that prion variants may be selected by changes in the chaperone network.     

Importance of the Hsp70 ATPase domain in yeast prion propagation

The Saccharomyces cerevisiae non-Mendelian genetic element [PSI+] is the prion form of the translation termination factor Sup35p. The ability of [PSI+] to propagate efficiently has been shown previously to depend upon the action of protein chaperones. In this article we describe a genetic screen that identifies an array of mutants within the two major cytosolic Hsp70 chaperones of yeast, Ssa1p and Ssa2p, which impair the propagation of [PSI+]. All but one of the mutants was located within the ATPase domain of Hsp70, which highlights the important role of regulation of Hsp70-Ssa ATP hydrolysis in prion propagation. A subset of mutants is shown to alter Hsp70 function in a way that is distinct from that of previously characterized Hsp70 mutants that alter [PSI+] propagation and supports the importance of interdomain communication and Hsp70 interaction with nucleotide exchange factors in prion propagation. Analysis of the effects of Hsp70 mutants upon propagation of a second yeast prion [URE3] further classifies these mutants as having general or prion-specific inhibitory properties.     

Chaperones that cure yeast artificial [PSI+] and their prion-specific effects

The [PSI(+)] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI(+)] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI(+)] against Hsp104 [3,4]. We used an artificial [PSI(+)(PS)] based on the Sup35 prion-forming domain from yeast Pichia methanolica [5] to find other prion-curing factors. Both [PSI(+)(PS)] and [PSI(+)] have prion 'strains', differing in their suppressor efficiency and mitotic stability. We show that [PSI(+)(PS)] and a 'weak' strain of [PSI(+)] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI(+)(PS)] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.     

A role for cytosolic hsp70 in yeast [PSI(+)] prion propagation and [PSI(+)] as a cellular stress

[PSI(+)] is a prion (infectious protein) of Sup35p, a subunit of the Saccharomyces cerevisiae translation termination factor. We isolated a dominant allele, SSA1-21, of a gene encoding an Hsp70 chaperone that impairs [PSI(+)] mitotic stability and weakens allosuppression caused by [PSI(+)]. While [PSI(+)] stability is normal in strains lacking SSA1, SSA2, or both, SSA1-21 strains with a deletion of SSA2 cannot propagate [PSI(+)]. SSA1-21 [PSI(+)] strains are hypersensitive to curing of [PSI(+)] by guanidine-hydrochloride and partially cured of [PSI(+)] by rapid induction of the heat-shock response but not by growth at 37 degrees. The number of inheritable [PSI(+)] particles is significantly reduced in SSA1-21 cells. SSA1-21 effects on [PSI(+)] appear to be independent of Hsp104, another stress-inducible protein chaperone known to be involved in [PSI(+)] propagation. We propose that cytosolic Hsp70 is important for the formation of Sup35p polymers characteristic of [PSI(+)] from preexisting material and that Ssa1-21p both lacks and interferes with this activity. We further demonstrate that the negative effect of heat stress on [PSI(+)] phenotype directly correlates with solubility of Sup35p and find that in wild-type strains the presence of [PSI(+)] causes a stress that elevates basal expression of Hsp104 and SSA1.     

Variant‐specific and reciprocal Hsp40 functions in Hsp104‐mediated prion elimination

The amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded proteins, passed to daughter cells following fragmentation by molecular chaperones including the J‐protein Sis1, Hsp70 and Hsp104. Overexpression of Hsp104 efficiently cures cell populations of the prion [PSI⁺] by an alternative Sis1‐dependent mechanism that is currently the subject of significant debate. Here, we broadly investigate the role of J‐proteins in this process by determining the impact of amyloid polymorphisms (prion variants) on the ability of well‐studied Sis1 constructs to compensate for Sis1 and ask whether any other S. cerevisiae cytosolic J‐proteins are also required for this process. Our comprehensive screen, examining all 13 members of the yeast cytosolic/nuclear J‐protein complement, uncovered significant variant‐dependent genetic evidence for a role of Apj1 (antiprion DnaJ) in this process. For strong, but not weak [PSI⁺] variants, depletion of Apj1 inhibits Hsp104‐mediated curing. Overexpression of either Apj1 or Sis1 enhances curing, while overexpression of Ydj1 completely blocks it. We also demonstrated that Sis1 was the only J‐protein necessary for the propagation of at least two weak [PSI⁺] variants and no J‐protein alteration, or even combination of alterations, affected the curing of weak [PSI⁺] variants, suggesting the possibility of biochemically distinct, variant‐specific Hsp104‐mediated curing mechanisms.     

Requirements of Hsp104p activity and Sis1p binding for propagation of the [RNQ+] prion

The formation and maintenance of prions in the yeast Saccharomyces cerevisiae is highly regulated by the cellular chaperone machinery. The most important player in this regulation is Hsp104p, which is required for the maintenance of all known prions.

The requirements for other chaperones, such as members of the Hsp40 or Hsp70 families, vary with each individual prion. [RNQ+] cells do not have a phenotype that is amenable to genetic screens to identify cellular factors important in prion propagation. Therefore, we used a chimeric construct that reports the [RNQ+] status of cells to perform a screen for mutants that are unable to maintain [RNQ+]. We found eight separate mutations in Hsp104p that caused [RNQ+] cells to become [rnq-]. These mutations also caused the loss of the [PSI+] prion. The expression of one of these mutants, Hsp104p-E190K, showed differential loss of the [RNQ+] and [PSI+] prions in the presence of wild type Hsp104p. Hsp104p-E190K inefficiently propagated [RNQ+] and was unable to maintain [PSI+]. The mutant was unable to act on other in vivo substrates, as strains carrying it were not thermotolerant. Purified recombinant Hsp104p-E190K showed a reduced level of ATP hydrolysis as compared to wild type protein. This is likely the cause of both prion loss and lack of in vivo function. Furthermore, it suggests that [RNQ+] requires less Hsp104p activity to maintain transmissible protein aggregates than Sup35p. Additionally, we show that the L94A mutation in Rnq1p, which reduces its interaction with Sis1p, prevents Rnq1p from maintaining a prion and inducing [PSI+].